Part:BBa_K2052015
ButCoat with strong RBS
Usage & Biology ( Description )
This composite part consists of RBS , a coding region ( ButCoAT ) and enables E.coli to secrete ButCoAT.
ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) is given down below :
Figure 1: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA
Figure 2: Crystal Structures of Acetobacter aceti Succinyl (Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
Cloning
Gel Results
Figure 1. We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane RBS ligated ButCoat (K2052018) was loaded with its single digested version. Digested ones gave a sharp lane at 3600 bp as we expected.
Characterization
Ligated Parts Transformation Results
Figure: After ligating ButcoaT and RFP, we have obtain colonies 2:1 (insert:vector) ratio, and transform them into E.coli BL21.
Confirmational PCR Result
Figure: Constitutive promoter-RBS-ButCoaT-GFP-Double terminator cloned into pSB1C3 and after ligation we have transformed into E.coli BL21. Colony PCR results here you can see; one primer stick on insert and another stick on vector. The expected lenght of product is around 850 bp.
Flowcytometry Result
After checking with confirmational PCR we have tried to validate RFP fluoresence experimenttaly through Fluorescence Microscope, Here you can see the analysis.
Figure: After measuring RFP signals with channel FL3 we have obtained these graphs. Here again upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only ButCoaT. Middle triplet stand for only RFP expressed bacterial culture analysis which gave a peak that could be used as a control group. The bottom triplet was RFP tagged ButCoaT expressed bacterial group and as we have shown there is any signal depend on RFP activity.
Since ButCoAT was tagged with RFP, to test whether it is functional or not, we have used fluoresence microscopy technique to show its expression is properly done or not. However, because ButCoAT doesn’t have a stop codon at the end it fused with RFP and they both become unfunctional from this fusion. Therefore, we could not see any peak.
Design Notes
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
For ButCoaT ; E.Coli is synthesised suitable for Codon Bias. In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.
Modelling
The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.
The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.
Source
The ribosome binding site (B0034) that we’ve used is gotten from iGEM part library. Source of ButCoaT : Roseburia intestinalis L1-82
References
Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases
Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in ButyrateProducing Bacteria from the Human Large Intestine
Sequence : http://www.uniprot.org/uniprot/C7GB37?sort=score
Pathway : http://www.genome.jp/dbget-bin/www_bget?reaction+R00393+R01179+R01359+R07832
Picture : http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
For ButCoaT ;
E.Coli is synthesised suitable for Codon Bias. In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.
Source
The ribosome binding site (B0034) that we’ve used is gotten from iGEM part library. Source of ButCoaT : Roseburia intestinalis L1-82
References
Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases
Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in ButyrateProducing Bacteria from the Human Large Intestine
Sequence : http://www.uniprot.org/uniprot/C7GB37?sort=score
Pathway : http://www.genome.jp/dbget-bin/www_bget?reaction+R00393+R01179+R01359+R07832
Picture : http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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